Embryonic stem cells are pluripotent cells capable of both proliferation in cell culture as well as differentiation towards a variety of lineage restricted cell populations that exhibit multipotent properties (Odorico et al., (2001) Stem Cells 19:193-204). Human embryonic stem cells are thus capable of commitment and differentiation to a variety of lineage-restricted paths resulting in very specific cell types that perform unique functions. Multipotent stem cells of mesodermal origin during development give rise to bone, cartilage, tendon, muscle and fat (Minguell et al., (2001) Exp. Biol. Med. 226:507-520). Postnatally, these same cells are termed mesenchymal stem cells (MSCs) and can be found in relative abundance in bone marrow. Since MSCs by definition retain a self-renewing property, they can be isolated and expanded in culture. Researchers have found that intravenous injection of these cells results in the presence of long lasting precursor cells in various tissues capable of restoring the levels of specific stem cell precursors depleted as a result of aging or disease. As such, MSCs represent a potential mechanism for gene therapy (Bianco et al., (2001) Stem Cells 19:180-192). More importantly, it has been found that implantation of these cells in various animal model systems leads to the differentiation of these cells at localized sites and the subsequent regeneration of tissues such as muscle, tendon, cartilage and bone.
One source of multipotent stem cells that can be expanded in culture and utilized locally for potential bone regeneration, bone healing and fracture repair is bone marrow-derived MSCs (Bruder et al., (1994) J. Cell Biochem. 56:283-294). Studies of this nature using bone marrow-derived MSCs have been carried out in animal models but have not been conducted in humans perhaps because of some inherent limitations in the use of MSCs. For example, despite the capacity to expand these cells in vitro, MSCs represent a rare subpopulation of cells found in the bone marrow. Thus, the isolation of bone-derived MSCs requires extensive purification.
Other limitations encountered when using this population of MSCs is that they are significantly heterogeneous. For example, there is extensive variability in cell expansion capabilities, some exhibiting the ability to undergo numerous cell doublings whereas others have a much reduced capacity (Minguell et al., (2001)). Expanded MSC populations can also exhibit features of senescence and apoptosis. Other limitations of these cells originate from the variability in the human sources from which they are derived. For example, MSC populations derived from bone marrow populations are influenced by a variety of factors such as sex, age and physiological condition of the donor.
An alternative source of MSCs with perhaps less inherent limitations is believed to be embryonic stem cells (ES cells). ES cells are now routinely prepared from a variety of species including rodents, primates and man. Techniques for isolating stable cultures of human ES cells have recently been described by Thomson et al., in U.S. Pat. No. 5,843,780 and J. Thomson et al., (1998) Science 282:1145-1147, both of which are hereby incorporated by reference in their entirety.
Generally, ES cells are highly homogeneous, exhibit the capacity for self-renewal, and have the ability to differentiate into any functional cell in the body. This self-renewal property can lead under appropriate conditions to a long-term proliferating capability with the potential for unlimited expansion in cell culture. Furthermore, it is understood, that if human ES cells are allowed to differentiate in an undirected fashion, a heterogeneous population of cells is obtained expressing markers for a plurality of different tissue types (WO 01/51616; Shamblott et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:113). These features make these cells a unique homogeneous starting population for the production of cells having therapeutic utility.
There have been efforts by researchers in the field to use mouse ES cells to expand and differentiate into numerous cell lineages and restore depleted cell populations in rodent models. Specifically, mouse ES cells have been expanded and differentiated via the formation of embryoid bodies into cells capable of expressing bone osteoblast marker genes and forming mineralized bone in vitro (Dani et al., (1997) J. Cell Sci. 110:1279-85).
Also, others have reported differentiation of bone marrow-derived human mesenchymal stem cells (hMSC) into cells of more than one connective tissue type, such as bone, cartilage, tendon, ligament, and dermis (see U.S. Pat. No. 5,486,359). Other systems have been proposed for regeneration and augmentation of bone using mesenchymal stem cells, combined with a ceramic material or resorbable biopolymer as disclosed in International patent publication WO 97/40137.
Furthermore, pluripotent stem cell populations have been isolated from human blastocysts such that 30% of the cells are derived from mesenchymal cells and can be identified by expression of osteonectin or osteocalcin (see U.S. Published Application No. 20030036194). To our knowledge, however, these ES cell-derived populations are not homogenous MSC populations and have not effectively enhanced bone formation in vivo. Accordingly, a significant challenge in using human ES cells for therapeutic purposes, or for studying particular cell types in vitro, has been to obtain cell populations that include a substantial subpopulation that is relatively uniform in characteristics.
It is noted that none of the above reviewed publications teach or suggest a method for deriving a substantially homogenous population of mesenchymal stem cells from human ES cells. Also, it is unclear whether any of the cell preparations exemplified in the art can be produced in sufficient quantities for mass marketing as a therapeutic composition for use in bone repair. Therefore, to realize the potential of human embryonic stem cells in managing human health and disease, it would be useful to develop novel methods for efficiently isolating a substantially homogenous population of mesenchymal stem cells from human ES cells for production of therapeutically important tissue types.